Heteropolypeptide



HETEROPOLYPEPTIDE Walter A. Darlington and Harold I. Weingarten, Dayton, Ohio, assign'ors to Monsanto Chemical Company, St. LOUIS, M0., a corporation of Delaware No Drawing. Filed Nov. 2, 1956, Ser. No. 619,965

3 (Claw-1'12) with the balance being substantially equimol'ar proportrons of the recurring DL-leucyl unit:

- -NHCH( J H2CH( a)2 and the recurring D-L-lysyl hydrohalide unit:

in which X is a salt-forming anion, the average molecular \lavgioglt of said heteropolypeptid'e being from 1,500 to For convenience, since the present h'eteropolypep'tides are made up of three different recurring units, they will be hereinafter referred to as terpolymers.

The present terpolymers are prepared by a condensation reaction, involving carbon dioxide cleavage, of the N-carboxy-anhydrides of DL-al'anine and DL-leuci-ne and the N-carboxy-anhydride of e-carbobenzyloxy-DL-lysine to give a terpolymer consisting of DL-alanyl, DL-leucyl, and E-carbobenzyloxy-DL-lysyl units, subsequent removal of the carbob'enzyloxy radical by treatment with a hydrohalide, and recovery of the hydrohalide of a DL- alanyl-DL-leucyl-DL-lysyl heteropolypeptide. Advantageously, we dissolve from substantially l to 4 moles of N-carboxy-DL-alanine anhydride, substantially 1 mole of N-carboxy-DL-leucine anhydride' andsubstantially 1 mole of the e-ca-rbobenzyloxy-N-carboxy DL-lysineanhydride in an inert solvent, add to the resulting solution a small quantity, say from 0.01% to 2.0%, of a polymerization initiator comprising a compound having a labile hydrogen atom, and allow the resulting reaction mixture to stand at ordinaryor increased-temperaturesunnited Sta s Patent til formation of a terpolymer consisting of DL-alanyl,

evolution of carbon dioxide, completion of the reaction may also be determined by noting cessation of the evolution of this by-product.

The media in which the polymerization is efiecte'd are generally inert solvents, i.e., liquids which dissolve the anhydrides at the reaction temperature and which do not react with either the anhydrides or the terpolymer. Suitable solvents are, e.g., dioxane, nitrobenzene, xylene, benzene, dimethylformamide, acetone, chloroform, carbon tetrachloride, chlorobenzene, trichloroethylene, cyclohexanone, ethyl acetate, etc. As will be appreciated by those skilled in the art, the nature of the solvent is a factor to be considered in arriving at the temperature conditions which are to be employed, e.g., obviously the use of ether as a solvent will require operation at substantially low temperatures.

Although formation of the DL-alanyl-DL-leucyl-e-carbobenzyloxy-DL-lysyl terpolymer will take place thermally, i.e., by simply heating a solution of the three N- carboxy anhydrides, I have found that the average molecular weight of the product can be better controlled and the reaction time can be substantially shortened by operating in the presence of a polymerization initiator com-' prising a basic compound having a labile hydrogen atom, for example, ammonia and any primary or secondary straight chained or cyclic amine such as ethylamine, butylamine, allylamine, dimethylamine, chloroethylamine, fi phenethylamine, benzylamine, aniline, p-naphthylamine, N-ethylaniline, sarcosine dimethylamide, diethylened-iamine, etc. Pyridine and triethylarnine containing water as a trace impurity may also be used. Other compounds having a labile hydrogen such as alcohols, phenols and thiols may be employed as polymerization initiators, but the amines are preferred because their use does not lead to possibly undesirable side-reactions.

The molar ratio of DL-alanyl units to the DL-leucyl and e-carbooenzyloxy-DL-lysyl units in the terpolymer is determined by the reactant proportions employed. Generally, the use of a 1:1:1 molar ratio mixture of the three N-carboxy anhydrides yields a terpolymer in which the DL-alanyl, DL-leucyl and the E-carbobenz'yloxy-DL- lysyl units are present in a 1:121 ratio, and the use of a 321:1 N-carboxy-DL-alanine anhydride-N-carboxy-DL- leucine anhydride-e-carbobenzyloxy-N-carboxy-DL-lysine anhydride mixture gives a terpolymer in which the DL- alanyl', DL-leucine and e-carbobenzyloxy-DL lysyl units are present in a 3:1:1 molar ratio. When it is desired to prepare a terpolymer in which two-thirds of the chain structure consists of DL-alanyl units with the balance being equimolar proportions of recurring DL-leucyl and e-car-b'obenzyloxy-DL-lysyl units, a 4:1:1 molar ratio of the N-carboxy-DL-alanine, N-carboxy-DL-lencine and ecarbobenzyloxy-DL-lysine anhydrides is used.

The carbobenzyloxy radical may be readily cleaved from the terpolymer by treating it with a hydrohalide, before or after separating it from the initial reaction mixture. The carbobenzyloxy-containing terpolymer may be recovered from the reaction mixture in which it was formed by filtration when the product is aprecipitate or by distilling off the solvent if the product be soluble in the initially employed solvent. The recovered carbobenzyloxy-containing terpolymer is then subjected to cleavage by mixing it with the hyd-rohalide, preferably with an anhydrous solution of the hydrohalide in an inert solvent such as glacial acetic acid, dry ether or dioxane, etc. In many instances, however; the carbob'enzyloxy polymer need not be separated from the initial give substantially theoretical yields of the hydrohalide, particularly when hydrogen bromide is the halide used. I-Iowever, other hydrogen halides, e.g., hydrogen chloride or hydrogen iodide may be used, whereby there is formed in the cleavage step the heteropolypeptide hydrochloride or hydroiodide. Salts other than the hydrobromides may also be conveniently prepared from the corresponding heteropolypeptide hydrobromide by treating the hydrobromide with a salt of a metal which forms diflicultly soluble bromides, removing the metal bromide thus formed and treating the residue with the desired acid, e.g., benzoic acid, acetic acid, tartaric acid, picric acid, phosphoric acid, perchloric acid, hydrochloric acid, etc., whereby there are formed, e.g., the corresponding DL- alanyl-DL leucyl-DL-lysyl heteropolypeptide benzoates, acetates, tartrates, etc. Useful metal salts for this purpose are, e.g., silver acetate or phosphate, etc.

The present DL alanyl, DL-leucyl-DL-lysyl heteropolypeptide salts are stable, well-defined powdery solids which are generally soluble in water, and insoluble in ethanol, ether, acetone, dioxane and chlorobenzene. They are advantageously employed for a variety of purposes in industrial, agricultural and medicinal fields, e.g., as anti-static agents for textiles and other fibrous products, as emollient bases, as plant growth regulants and as bactericides. The present terpolymer salts are particularly valuable in that they inhibit the multiplication and growth of viruses after infection has occurred, especially those which cause mosaic diseases in tobacco.

Although a great deal of research has been directed at tobacco mosaic virus control, the search for chemicals that will interfere with or inactivate tobacco mosaic virus after it has been established in the tobacco plant has not been at all fruitful. While a number of compounds when mixed with infectious: tobacco mosaic virus in vitro form non-infectious complexes with it (F. C. Bawden, Plant Viruses and Virus Diseases, Chronica Botanica, Waltham, Mass, 1950, p. 244), the infectivity of the virus is frequently restored after dilution. Hence successful in vitro experiments rarely foretell success in treatment of plants by spraying or dusting already infected plants.

The invention is further illustrated, but not limited, by the following examples:

Example 1 A mixture consisting of 5 g. (0.0435 mole) of the N-carboanhydride of DL-alanine, 2.28 g. (0.015 mole) of the N-carboanhydride of DL leucine and 4.45 g. (0.015 mole) of the N-carboanhydride of e-carbobenzyloxy DL-lysine was dissolved in 250 ml. of dry dioxane. A solution of diethylamine was prepared by adding 0.28 g. of the amine to 550 ml. of dry dioxane. To this amine solution there was then added 50 ml. of said dioxane solution of anhydrides, and subsequent 50 ml. portions of the anhydride solution were added to the amine solution every hour thereafter until all of the anhydride solution had been used. The whole was then allowed to stand overnight for a total reaction time of about 22 hours. The precipitate which had formed in the reaction mixture was filtered off and dried to give 5.9 g. (69% theoretical yield) of terpolymer wherein substantially three-fifths of the polymer chain consists of recurring DL units, with the balance being equimolar proportions of recurring DL-leucyl and recurring ecarbobenzyloxy-DL-lysyl units.

It was labelled Fraction I. A second fraction of copolymer was obtained by treating the filtrate with an excess of dilute hydrochloric acid, and collecting and drying the precipitate which formed. The quantity of Fraction II thus obtained amounted to 1.2 g. (14% theoretical yield).

End group analysis of Fraction I showed an average of 27 amino acid residues per polymer molecule and a consequent average molecular weight of 3,100. End

group analysis of Fraction 11 showed an average of 76 amino acid residues per polymer molecule, which corresponds to an average molecular weight of 8,750.

The carbobenzyloxy radical was cleaved from the Fraction I terpolymer as follows: 5.4 g. of the polymer was treated with 20 -g. of 33% solution of hydrogen bromide in glacial acetic acid, 25 ml. of acetic was added to' the resulting mixture and the whole was allowed to stand overnight. At the end of that time ml. of dry ether was added to the mixture and the precipitate which formed was collected, vacuum-dried and ground to give 5.25 g. of the powdery heteropolypeptide wherein substantially three-fifths of the polymer chain consists of recurring DL-alanyl units with the balance being substantially equimolar proportions of DL- leucyl and DL-lysyl hydrobromide units.

Example 2 The inhibiting efiect of the 3:121 DL-alanyl-DL- leucyl-DL-lysy-l hydrobromide polypeptide of Example 1 against the multiplication of tobacco mosaic virus was determined substantially accordang to the method of Commoner et al., Arch. Biochem. Biophys, 27 271 (1950). Briefly, the procedure involves inoculation of a young leaf of a healthy plant with the virus and, after 24 hours, contact of portions of the inoculated leaf with the test compound.

The leaf was inoculated with Johnson tobacco virus by rubbing its entire surface with a gauze pad moistened with a phosphate buffer solution (pH 7.0) containing 200 ,ug. of the virus per milliliter. After inoculation the leaf was placed under a bell-jar with its petiole in water for 24 hours. At the end of this time, siX 0.5" discs were punched from the leaf, weighed, washed in water and placed in a 3.5" Petri dish containing an 0.0067% concentration of the said test heteropolypeptide in 15 ml. of half-strength Vickerys solution (Vickery et al., Bull. Conn. Agr. Expt. Sta., 399 (1937)) which had been prepared to have a final concentration of 5 10- mole of KI-I PO A control was prepared by placing another set of six discs which had been punched from the inoculated leaf into a Petri dish containing 15 ml. of the same Vickerys solution but no terpolymer. Both dishes of discs were incubated for seven days under fluorescent light of foot candles. At the end of that time the discs were removed and two groups of three were made up from each dish in order to provide checks. The tobacco mosaic virus content of each group was determined as described by Commoner et al. (loc. cit.) except that the final washing step thereof was omitted because it had been previously observed by us that this step in the isolation procedure results in some loss of virus. The colorimetric measurements were made at 750 ITl/.L in a Coleman Universal Spectrophotometer and the amount of virus was read from a standard curve prepared with known amounts of tobacco mosaic virus.

Employing this procedure it was found that the virus content of the group of discs which had been contacted with the present DL-alanyl-DL-leucyl-DL-lysyl terpolymer was only 35% of that of the control discs, i.e., a 65% inhibition of the virus was effected by the present terpolymer.

The marked inhibition of tobacco mosaic virus growth by the present terpolymer is surprising, for generally polypeptides having alanyl, leucyl or lysyl units do not demonstrate this effect. Thus, using the above-described testing procedure the following terpolymers were found to have no inhibiting effect on tobacco mosaic virus:

(I) 3:1:1 DL-alanyl-DL-phenylalanyl-DL-lysyl hydrobromide, mol. wt. 2900.

(II) 32121 DL-alanyl-DL-valyl-diaminobutyryl hydrobromide, mol. Wt. 4400.

(III) 3:1:1 DL-alanyl-aminoisobutyryl-DL-lysyl hydrobr m de 2 91.- WL 26 0.

5 (IV) 3:1:1 DL-alanyl-DL-etbionyl-DL-lysyl hydrobromide, mol. wt. 3560.

(V) 3:1:1 DL-alanyl-DL-methionyl-DL-lysyl hydrobromide, mol. wt. 3460.

(VI) 3:1:1 DL-alanyl-DL-tryptophanyl-DL-lysyl hy- 5 drobromide, mol. wt. 4030.

(VII) 3:1:1 Glycyl-DL-valyl-DL-lysyl hydrobromide,

Y mol. wt. 6500.

(VIII) 1: 1 1 1 Glycyl-DL-alanyl-DL-valyl-DL-lysyl hydrobromide, mol. Wt. 6550.

The above shows that the presence of any one of the present terpolymer components, i.e., the alanyl, leucyl or lysyl unit, in a polypeptide does not necessarily lead to virus inhibiting effect. Apparently all three components are required. Note, for example that in (I) above, the 15 terpolymer resembles the Fraction I of Example 1 which was tested in that the DL-alanyl and the DL-lysyl radical are present and that the molecular weights are of the same order. Apparently, the presence of the DL-phenylalanyl unit instead of the DL-leucyl unit results in a ter- 20 polymer with no inhibiting efiect at the same test concentration. That no inhibition is obtained when the DL-leucyl component is changed is also shown in (III)(VI) above. On the other hand in (II), the replacement of only the lysyl unit likewise appears to be disadvantageous, and in VII, the replacement of only the alanyl unit also gives a terpolymer which has no effect against the virus.

Heteropolypeptides consisting of DL-alanyl, DL-leucyl and DL-lysyl units and a molecular weight of from 1,500 3 in which X is halogen, said unit (I) constituting from one-third to about two-thirds of the terpolymer, with the balance of the terpolymer being the units (111) and (H) in about equal amounts.

6 2. A condensation terpolymer having an average molecular weight of from 1,500 to 10,000 and containing in the terpolymer chain recurring units of the formulas in which X is halogen, said unit (I) constituting about three-fifths ocf the terpolymer with the balance of the terpolymer being the units (III) and (II) in about equal amounts.

3. A condensation terpolymer having an average molecular weight of from 1,500 to 10,000 and containing 5 in the terpolymer chain recurring units of the formulas said unit (I) constituting about three-fifths of the terpolymer with the balance of the terpolymer being the units (III) and (II) in about equal amounts.

References Cited in the file of this patent UNITED STATES PATENTS 2,273,849 Epstein et al. Feb. 24, 1942 2,471,339 Minich et a1. May 24, 1949 2,498,665 Emerson Feb. 28, 1950 2,723,973 Boothwyn et a1. Nov. 15, 1955 OTHER REFERENCES Anson: Advances in Protein Chemistry, vol. 6, pages 136-45 (1951).

Tsuyuki et al.: Proceedings of the Soc. for Experimental Biology and Medicine, vol. 91, page 318, Jan. 9, 1956.

Sela et al.: J. Am. Chem. Soc., vol. 78, page 746, Feb. 20, 1956. 

1. A CONDENSATION TERPOLYMER HAVING AN AVERAGE MOLECULAR WEIGHT OF FROM 1,500 TO 10,000 AND CONTAINING IN THE TERPOLYMER CHAIN RECURRING UNITS OF THE FORMULAS 